Perturbation of energy metabolism via NAD supplementation modifies the expression of larval development time, a genetically based trait relevant to development and whole-organism fitness in Drosophila melanogaster. The results suggest a link between energy metabolism and gene expression. Supplemental NAD significantly decreased larval development time for isofemale strains chosen on the basis of development time differences. This effect did not represent a trade-off with larval survival, and was not related to the effects of supplemental glucose. The isofemale strains were assayed by using a method derived from Warburg's manometric procedures which measured changes in the proportions of ATP, ADP and AMP in larval homogenates. Supplemental NAD appeared to increase the proportion of ATP and the [ATP]/[ADP] ratio in vitro and during larval development. The decrease in larval development time mediated by NAD was attributed to supplemental NAD modifying the NAD pool beyond levels established during development, directly altering electron transport chain activity, ATP, ADP and AMP levels, and possibly gene expression.